ORIGINAL ARTICLE

The article was published by Academy of Chemistry of Globe Publicationswww.acgpubs.org/RNP © Published 03/25//2012 EISSN:1307-6167

Rec. Nat. Prod. 6:3 (2012) 242-254

Synthesis and Myocardial Ischemia Protective Effect of

Ocotillol-Type Derivatives

Bi Yi1, Wang Tian2, Meng Qingguo1 *, Zhang Jiangfen1, Wang Liang1, Li

Qiang2, Zhao Fenglan1 and Sun Haijun1

1Department of Medicinal Chemistry, School of Phamacy, Yantai University, 32 Qingquan Road,

Yantai 264005, China2 Department of pharmacology，School of Phamacy, Yantai University, 32 Qingquan Road, Yantai

264005, China

(Received March 12, 2011; Revised November 8, 2011; Accepted December 11, 2011)

Abstract: A series of ocotillol-type derivatives bearing a glycosyl moiety were synthesized. Among them,compound 3a and 3b led to a reduction in the increased CK, LDH, MDA activity and attenuate the decrease ofSOD, GSH-PX and T-AOC compared with the iso group. In the heart section, compound 3a and 3b groupshowed nearly normal myofibrillar structure nearly without neutrophil granulocytes infiltration in the iso group.These results indicated that ocotillol-type derivatives may be the good lead compounds with protective propertyof myocardial ischemia.

Keywords: Ocotillol-type derivatives; myocardial ischemia; synthesis; ginsenoside.

1. Introduction

Ginseng (Panax ginseng C.A. Meyer) have been used in China for about 1000 years for thetreatment of cardiovascular diseases and stroke. Ginsenosides are considered to be the main bioactiveconstituents. It is reported that gensenoside F11 and ocotillol derivatives have the potential clinicalvalue in myocardial ischemia therapy.[1,2] In our previous study, we have obtain the ocotillol-typesaponin and derivatives and proved their cardioprotective effects on myocardial injury induced by ISOin rats.[3] The source of octillol-type gensenoside is fewer than other type in natural world. So wedesign and synthesize a series of derivatives of octillol from 20(S)-Rh2, 20(S)-Rh1 and report theirprotective effect in myocardial injury induced by isoproterenol in rats.

*Corresponding author: E-Mail: qinggmeng@163.com; Tel: +86-535-6706022, Fax: +86-535-6706066

Yi et.al., Rec. Nat. Prod. (2012) 6:3 242-254243

2. Materials and Methods

2.1. Material

The material 20(S)-Rh2 and 20(S)-Rh1 were degraded and isolated from the crude product ofginsides in our laboratory (Rh1. m.p 191-194℃, lit. 192-194℃. Rh2. m.p 218-221℃, lit. 218-220℃).[4] The other reagents were bought from different companies in China. Melting points were determinedusing a digitzing melting point apparatus (WRS-1B) and were not corrected. All of the compoundssynthesized were purified by column chromatography (CC) on silica gel (200-300 mesh) andthin-layer chromatography (TLC) on silica gel GF254 plates (Yantai Chemical Industry ResearchInstitute, China). Subsequently, they were routinely analyzed by 1H-NMR (Bruker VANCE-400), MS(Applied Biosystoms Mariner spectrometer).

2.2. Synthesis

(3β,12β)-12-acetyl-20-hydroxydammar-24-en-3-yl-β-D-(2’,3’,4’,6’-tetra-acetyl)-glucopyranoside(1a). 20(S)-Rh2 (4.92g, 7.91mmol) and acetic anhydride (54.3ml, 47.25mmol) were dissolved inpyridine (55ml) and stirred for 2h in room temperature. After evaporation in vacuum and the residuewas taken up in ethyl acetate and dilute hydrochloric acid. The organic phase was separated andwashed with water and saturated sodium chloride solution, dried (Na2SO4), and then concentrated toyield a semi-solid. Flash chromatography (1:1 petroleum ether-ethyl acetate) gave the compound 1a asa white solid (4.03 g, 62.0% yield, m.p 221-222℃, lit 223-225℃ (form EtOH)[5]). The results of13C-NMR are shown in Table 1.

(3β,12β)-12-acetyl-20S,24R-epoxy-dammar-24-en-3-yl-β-D-(2’,3’,4’,6’-tetra-acetyl)-glucopyranoside (2a). A solution of compound 1a (4.03 g, 4.90 mmol) in dichloromethane (30ml) was cooled to-3℃. Then a solution of m-CPBA (0.95 g, 5.50 mmol) in dichloromethane (10 ml) was added slowlyand stirred for 1 h. The organic solution was washed with water and saturated sodium chloridesolution and dried over Na2SO4. The dichloromethane was evaporated in vacuo to yield a white solid.The residue was chromatographed over silica gel (1:1 petroleum ether-ethyl acetate) and crystallizedfrom ethyl acetate to get compound 2a as white needle-like crystals (2.90 g, 73.2% yield, m.p203-204℃). The results of 13C-NMR are shown in Table 1.

(3β,12β)-12-hydroxy-20S,24R-epoxy-dammar-24-en-3-yl-β-D—glucopyranoside (3a)[6]. Sodiumhydroxide (2.00g, 50.00mmol) was added to the solution of compound 2a (2.90 g, 3.58 mmol) inmethanol (30 ml). The resulting mixture was stirred at room temperature for 2 h. The solvent wasdiluted with water 200 ml. After filtration, the residue was chromatographed over silica gel (ethylacetate) and crystallized from ethyl acetate yielded compound 3a as white pellet-like crystals (0.75g,33.2% yield, m.p 184-185℃). HRMS for C36H62O9 + H calcd 639.44666, found 639.44883.ESI-MS,m/z: 639.37[M + H ]+, 661.2[ M + Na ]+. 1H-NMR (C6D5N, 400 Hz) δ: 0.75 0.91 0.97, 1.24, 1.27，1.30, 1.30，1.45 (each 3H, s), 3.36 (1H, m) 3.70 (1H, m), 3.93 (1H, t, J=7.96Hz), 4.04 (1H, m), 4.25(2H, m), 4.42(1H, t, J=5.36Hz), 4.60 (1H, m), 4.84 (1H, m), 4.94 (1H, d, J=7.76Hz). The results of13C-NMR, HMBC, HSQC, 1H-1H COSY are shown in Table 2.

(3β,6α,12β)-3,12-diacetyl-20-hydroxy-dammar-24-en-6-yl-β-D-(2’,3’,4’,6’-tetra-acetyl)-glucopyranoside (1b). According to the procedure for the preparation of 1a, treatment of 20(S)-Rh1 (9.25g,14.50mmol) afforded 1b as a white powder (8.88g, 68.1% yield, m.p 185-186℃). The results of13C-NMR are shown in Table 3.

(3β,6α,12β)-3,12-diacetyl-20S,24R-epoxy-dammar-24-en-6-yl-β-D-(2’,3’,4’,6’-tetra-acetyl)-glucopyranoside (2b). According to the procedure for the preparation of 2a, treatment of 1b (8.88g,9.86mmol) afforded 2b as white solid (4.52g, 50.1% yield, m.p 187-188℃). The results of 13C-NMRare shown in Table 3.

Myocardial ischemia protective effect of ocotillol-type derivatives 244

(3β,6α,12β)-3,12-dihydroxy-20S,24R-epoxy-dammar-24-en-6-yl-β-D-glucopyranoside (3b)[7].According to the procedure for the preparation of 3a, treatment of 2b (4.52g, 4.94mmol) afforded 3bas white needle-like crystals (1.12 g, 34.3% yield, m.p 224-225℃). HRMS for C36H62O10 + H calcd655.44157, found 655.44354. ESI-MS, m/z: 655.41 [ M + H ]+ , 677.39 [ M + Na ]+. 1H-NMR(C6D5N,400Hz) δ: 0.75 (3H, s, CH3-19)，1.00, 1.18, 1.24 , 1.24, 1.24, 1.27, 1.45 (each 3H, s), 3.52 (1H, d,J=10.28Hz), 3.68 (1H,m), 3.94 (1H, m), 4.08 (1H, d, J=7.48Hz), 4.23 (2H, m), 4.45 (3H, m), 4.82(1H, s), 5.01 (1H, m). The results of 13C-NMR, HMBC, HSQC are shown in Table 4.

2.3. Pharmaceutical Experiment

Except control group and isoproterenol alone group, rats in therapeutic groups were orally givenRh1（67.8 mg/kg），3a（69.5 mg/kg），Rh2（71.9 mg/kg），3b（73.6 mg/kg）once a day for 7 days,respectively. On the fifth day, except control the group, rats were subcutaneously injected withisoproterenol (20 mg/kg) once a day for 3 days. The experiment was stopped 2 h after the last injectionof isoproterenol, and rats were anesthetized intraperitoneally with urethane (1 g/kg). A blood samplewas drawn from abdominal aorta, centrifuged and the serum samples were assayed for LDH and CKactivities. Then the rats were killed, and hearts were removed rapidly. The MDA，SOD、GSH-Pxactivity and T-AOC content in left ventricle homogenates were measured according to the kitinstructions. In each group, two rat hearts was taken for pathological examination afterhematoxylin–esosin staining. [8,9]

3. Results and Discussion

The mild saponification method of Ginsenoside has been explored. Panaxadiol saponins andpanaxatriol saponins in DMSO were heated and degraded by treatment with sodium methoxide.20(S)-Rh2 and 20(S)-Rh1 were obtained from panaxadiol saponins and panaxatriol saponins.Synthesis of derivatives of 20(S)-Rh2, 20(S)-Rh1 is shown in Scheme 1 and two target compounds aresuccessfully obtained by acetylation, oxidation and saponification.

The structures of these ocotillol-type derivatives are elucidated by 1H-NMR, 13C-NMR, MS,DEPT, HMBC, HSQC or HRMS.

Isoproterenol is widely used as an agent to evaluate cardioprotective drugs and study myocardialconsequences of ischemic disorders. The pharmaceutical experiment results are shown in Fig 1-7. Theserum CK and LDH activity augmented in the Iso group as compared with that in the Con group.Administration with 3a and 3b led a reduction in the increased CK and LDH activity, respectively.The CK and LDH activity did not significantly decrease after treatment with 20(S)-Rh1, 20(S)-Rh2 in.After isoproterenol injection, the MDA levels increased in the heart tissues significantly. Treatmentwith 3a and 3b, MDA levels show a significant reduction in groups but not in Con group. MDA levelsin 20(S)-Rh1, 20(S)-Rh2 group did not cause decrease as compared with Iso group. SOD, GSH-PXand T-AOC in heart tissues showed a significant decrease in Iso group. Treated with 3a or 3battenuate the decrease of SOD, GSH-PX and T-AOC in the group. Administrated with 20(S)-Rh1,20(S)-Rh2 did not led a reduction of SOD, GSH-PX and T-AOC. The heart sections in Con groupshowed a normal myofibrillar structure with striations, branched appearance and continuity withadjacent myofibrils. In Iso group the heart sections revealed extensive myofibrillar degeneration,which is related to infiltration with neutrophil granulocytes and interstitial edema. Treatment with20(S)-Rh1, 20(S)-Rh2 group, the heart sections showed mild degenerative changes and discontinuitywith adjacent myofibril. In 3a and 3b group, the heart sections showed nearly normal myofibrillarstructure nearly without neutrophil granulocytes infiltration.

Yi et.al., Rec. Nat. Prod. (2012) 6:3 242-254245

OHCH3

R1

CH3OAc

R1

OH

R1

HOHO

O

CH3H3COH

R2 R2

R2

a

c

24

Rh2 R1=OO

HO

OHOH

OH

bOAc

R1

O

CH3H3COH

R2

24

Rh1 R1=OH R2= O

HO

OHOH

OH

O

R2=H

3a R1=OO

HO

OHOH

OH

3b R1=OH R2= O

HO

OHOH

OH

O

R2=H

1a R1=OO

AcO

OAcOAc

OAc

1b R1=OAc R2= O

AcO

OAcOAc

OAc

O

R2=H

2a R1=OO

AcO

OAcOAc

OAc

2b R1=OAc R2= O

AcO

OAcOAc

OAc

O

R2=H

Scheme 1 Reagents and conditions: a) (CH3CO)2O, DMAP. Pyridine; b) m-CPBA, CH2Cl2; c) NaOH, CH3OH,H2O

4. ConclusionThe study indicated that compound 3a and 3b had the protective effectt of myocardial ischemia

associated with their antioxidant activity. The octillol-type derivatives maybe the good leadcompounds with protective property of myocardial ischemia. Further biological evaluation of theseoctillol-type derivatives and extensive lead optimizations are ongoing in our laboratory and moreresults will be reported in due time.

Table.1 13C NMR data of compounds 1a and 2a（C6D5N, δ, ppm）

Myocardial ischemia protective effect of ocotillol-type derivatives 246

No. 1a 2a 20(s)-Rh21 39.8 39.8 39.42 27.6 27.8 27.33 91.0 91.1 88.94 40.0 40.0 40.35 57.2 57.2 56.46 19.2 19.2 18.77 34.5 35.7 36.08 37.9 38.0 37.29 51.2 51.1 50.710 39.8 39.8 39.811 32.3 32.2 32.212 71.0 70.1 71.113 48.6 48.6 48.814 51.2 51.1 51.915 31.4 32.2 31.516 26.9 26.9 26.817 53.7 53.4 54.818 16.7 16.7 16.819 16.6 16.5 16.420 73.2 84.7 73.221 26.9 27.1 27.022 35.6 35.6 35.423 23.4 23.3 23.124 126.2 84.7 126.425 132.0 70.1 130.726 25.7 25.9 25.727 17.7 17.8 17.728 28.2 28.3 28.329 16.0 16.1 16.030 17.1 17.8 17.3

3-glc-1’ 103.7 103.7 106.72 75.2 74.3 75.83 72.6 72.6 78.74 73.2 73.3 72.2

5 76.8 77.0 78.06 63.2 63.2 63.3

Table.2 NMR data for Compound 3aδc Correlated proton

No. 20(S-)Rh2 3a HSQC HMBC 1H-1H COSY

1 39.4 39.2 1.52(1e), 0.79(1a) 19 1e, 2a2 27.3 26.7 2.18(2e), 1.82(2a) 1a, 3, 2e3 88.9 88.7 3.36(dd, J= 4.4, 4.3

Hz)3-glc-1,28,29 2a, 2e

4 40.3 40.0 0.74 28,295 56.4 56.4 0.66(d, J=10.0 Hz) 28,29,19 6a6 18.7 18.4 1.51(6e), 1.39(6a) 5,7a7 36.0 35.1 1.37(7e), 1.24(7a) 18 6e, 6a

Yi et.al., Rec. Nat. Prod. (2012) 6:3 242-254247

8 37.2 36.9 18,9 50.7 50.0 1.80 18,19 11e10 39.8 39.7 1911 32.2 32.4 2.01(11e),

1.20(11a)30 9,13,12

12 71.1 71.0 3.75 (m) 11a, 1313 48.8 48.3 2.25(m) 21 11,12,1714 51.9 50.7 1.46 18,3015 31.5 31.6 1.87(15e),

1.60(15a)21 16

16 26.8 25.5 2.14(16e),1.86(16a)

17 17,15a

17 54.8 52.1 18,30 13,16

18 16.8 16.7 0.97(s) 7

19 16.4 16.5 0.75(s)

20 73.2 86.7 21

21 27.0 27.2 1.27(s)

22 35.4 32.8 1.92(22e),1.06(22a)

21 23a, 23e

23 23.1 28.1 2.16(23e),1.84(23a)

27 22a, 22e

24 126.4 85.6 3.93(dd, J =7.0, 7.0Hz)

26,27 23a, 23e

25 130.7 70.3 26,27

26 25.7 27.6 1.24(s)

27 17.7 26.9 1.45(s)

28 28.3 28.1 1.30(s) 29,3

29 16.0 15.5 0.96(s) 28,5

30 17.3 18.3 0.91(s)

3-glc-1’ 106.7 106.7 4.95(d, J =8.0 Hz) 3

2’ 75.8 75.8 4.04

3’ 78.7 78.8 4.23

4’ 72.2 71.9 4.25 3’

5’ 78.0 78.4 4.00 4’

6’ 63.3 63.1 4.59,4.62

Table.3 13C NMR data of compounds 1b and 2b（C6D5N,δ, ppm）

Myocardial ischemia protective effect of ocotillol-type derivatives 248

No. 1b 2b 20(S)-Rh11 39.24 39.22 39.382 27.49 27.97 26.553 76.34 76.66 78.304 40.41 40.41 40.065 61.34 61.31 61.146 77.80 76.66 77.837 45.41 45.43 44.958 41.88 41.83 40.819 50.51 50.37 49.50

10 39.01 39.22 39.1111 30.84 30.85 30.9412 69.72 69.70 70.7413 48.36 48.35 47.9614 51.20 50.37 51.3415 32.22 32.15 31.4316 26.00 26.21 26.7317 53.40 53.17 54.4718 17.28 17.27 17.3819 17.30 17.21 17.3420 72.75 87.00 72.7021 26.43 26.93 26.7322 35.60 35.6 35.5323 21.75 23.3 22.7024 126.21 84.80 126.0325 131.98 70.1 130.4826 25.88 25.9 25.5227 17.27 17.8 17.0928 32.21 28.3 31.7829 17.58 16.1 16.5030 17.68 17.8 16.08

6-glc-1’ 102.44 102.42 105.732 75.13 75.09 75.183 80.77 80.77 79.764 72.75 72.75 71.585 82.47 82.47 79.356 63.51 63.50 62.79

Table.4 NMR data for Compound 3b

Yi et.al., Rec. Nat. Prod. (2012) 6:3 242-254249

δc Correlated proton

NO. 20(S)- Rh1 3b HSQC HMBC1 39.28 39.56 1.45(m), 0.84(m) 192 26.55 27.89 1.74(m), 1.82(m) 193 78.30 78.54 3.47(dd, J= 7.5, 11.3 Hz) 28,294 40.06 40.35 28,295 61.14 61.50 1.39(d, J=11.0 Hz) 28,29,196 77.83 78.14 4.66(dd, J= 3.0, 9.5 Hz) 28,297 44.95 45.08 1.98,2.5 188 40.81 40.99 18,309 49.90 49.42 1.54(d,7.0) 18,1910 39.11 39.44 1911 30.94 32.4 1.93(m),1.98(m) 18,1912 70.74 71.16 3.69(m)13 47.96 48.37 1.54(m) 3014 51.34 52.13 1.46 18,3015 31.43 32.39 1.26(m), 1.60(m) 18,3016 26.73 25.40 1.45(m), 1.74(m)17 54.47 50.52 2.08(m) 2118 17.38 17.99 1.00(s)19 17.34 17.82 0.83(s) 28,2920 72.70 86.67 2121 26.73 27.66 1.27(s)22 35.53 32.68 1.92(m), 1.06(m) 2123 252.70 28.75 2.16(m), 1.84(m) 26,2724 126.03 85.58 4.16(dd, J =7.0, 7.0 Hz) 26,2725 130.48 70.27 26,2726 25.52 27.11 1.30(s) 2727 17.09 26.90 1.45(s) 2628 31.78 31.64 1.28(s) 29,1929 16.50 17.04 1.11(s) 2830 16.08 16.24 0.91(s)

6-glc-1’ 105.73 105.96 4.95(d, J =8.0 Hz) 32’ 75.18 75.44 4.043’ 79.76 80.01 4.264’ 71.58 71.89 4.25 3’5’ 79.35 79.63 4.00 4’6’ 62.79 63.12 4.56,4.39

Myocardial ischemia protective effect of ocotillol-type derivatives 250

Figure.1 Effects of 20(S)-Rh1, 20(S)-Rh2 and its derivatives on CK activity in isoproterenol-inducedmyocardial injury in rats. Data was expressed as the mean ± S.D. (n = 6 to 8). Statistical significances weredetermined using unpaired two-tailed Student t-test or one-way analysis of variance (ANOVA) followed byDunnett’s contrast. ##P<0.01 compared with control group; **P<0.01 compared with isoproterenol group.

Figure.2 Effects of 20(S)-Rh1, 20(S)-Rh2 and its derivatives on LDH activity in isoproterenol-inducedmyocardial injury in rats. Data was expressed as the mean ± S.D. (n = 6 to 8). Statistical significances weredetermined using unpaired two-tailed Student t-test or one-way analysis of variance (ANOVA) followed byDunnett’s contrast. #P<0.05 compared with control group; *P<0.05compared with isoproterenol group.

Figure. 3 Effects of 20(S)-Rh1, 20(S)-Rh2 and its derivatives on MDA in isoproterenol-induced myocardialinjury in rats. Data was expressed as the mean ± S.D. (n = 6 to 8). Statistical significances were determined usingunpaired two-tailed Student t-test or one-way analysis of variance (ANOVA) followed by Dunnett’s contrast.##P<0.01 compared with control group; *P<0.05 compared with isoproterenol group.

Yi et.al., Rec. Nat. Prod. (2012) 6:3 242-254251

Figure.4 Effects of 20(S)-Rh1, 20(S)-Rh2 and its derivatives on SOD activity in isoproterenol-inducedmyocardial injury in rats. Data was expressed as the mean ± S.D. (n = 6 to 8). Statistical significances weredetermined using unpaired two-tailed Student t-test or one-way analysis of variance (ANOVA) followed byDunnett’s contrast. ##P<0.01 compared with control group; *P<0.05 compared with isoproterenol group.

Figure.5 Effects of 20(S)-Rh1, 20(S)-Rh2 and its derivatives on GSH-PX activity in isoproterenol-inducedmyocardial injury in rats. Data was expressed as the mean ± S.D. (n = 6 to 8). Statistical significances weredetermined using unpaired two-tailed Student t-test or one-way analysis of variance (ANOVA) followed byDunnett’s contrast. ##P<0.01 compared with control group; *P<0.05, **P<0.01 compared with isoproterenolgroup.

Myocardial ischemia protective effect of ocotillol-type derivatives 252

Figure. 6 Effects of 20(S)-Rh1, 20(S)-Rh2 and its derivatives on T-AOC in isoproterenol-induced myocardialinjury in rats. Data was expressed as the mean ± S.D. (n = 6 to 8). Statistical significances were determined usingunpaired two-tailed Student t-test or one-way analysis of variance (ANOVA) followed by Dunnett’s contrast. #P<0.05 compared with control group; *P<0.05 compared with isoproterenol group.

A B

C D

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E F

Figure.7 Effects of 20(S)-Rh1, 20(S)-Rh2 and its derivatives on histological change in isoproterenol-inducedmyocardial injury in rats.A. Haematoxylin and eosin stained heart section of control group (×200).B. Haematoxylin and eosin stained heart section of Iso group (×200).C. Haematoxylin and eosin stained heart section of 20(S)-Rh1 group (×200).D. Haematoxylin and eosin stained heart section of 3a group (×200).E. Haematoxylin and eosin stained heart section of 20(S)-Rh2 group (×200).F. Haematoxylin and eosin stained heart section of 3b group (×200).

Acknowledgments

The authors are grateful to National Natural Science Foundation of China (No.81001358), AProject of Shandong Province Higher Educational Science and Technology Program (No.BS2010YY073)，Scientific Reasearch Foundation of the Higher Education Institutions of ShandongProvince, China (No. J07WE26), Shandong Provincial Natural Science Foundation, China （No.Y2007C138） for financial support. We also gratefully acknowledge Mr. Jiyong Chen and Ms. LiShen for the measurement of ESI-MS and NMR and Mr. Guangzhi Shan for HRMS

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